Differential Interference Contrast (DIC) Microscopes
A major problem of imaging transparent specimens with conventional microscopes is that it can be difficult to elicit contrast, because the imaging technique is solely based on the amplitude information of the sampling light. This difficulty can be especially problematic for examining transparent or nearly transparent biological specimens. Phase information, if measured, can improve the imaging contrast dramatically. Conventional DIC microscopy performs admirably in this respect by rendering excellent phase contrast in these biological specimens, and is widely used in biology and clinical laboratories.
DIC microscopes are beam-shearing interference systems. An example of a conventional DIC microscope can be found in Murphy, Schwartz, Salmon, Spring, Parry-Hill, Sutter, and Davidson, “Differential Interference Contrast (DIC), 2007,” available from Nikon MicroscopyU at http://www.microscopyu.com/articles/dic/dicindex.html, which is hereby incorporated by reference in its entirety for all purposes. In DIC microscopes, a reference beam is sheared by a very small distance with respect to a sample beam. The phase difference between the reference beam and the sample beam after they pass two adjacent spots of the specimen provides the differential phase contrast of the specimen. Since DIC microscopy is an interference-based technique, it can distinguish minuscule amounts of phase differences and identify small changes in the sample's refractive index.
Prior art DIC microscopes have some disadvantages. Firstly, prior art DIC microscopes are very expensive instruments, as many complicated and expensive optical components are required to manipulate the light. Secondly, the lateral resolution of current DIC microscopes is determined by the spot size of the objective lens of the DIC microscope, which has a diffraction limit. The small sheared distance between the reference beam and the sample beam is usually tuned to be slightly smaller than this spot size.
Microfluidics
Recent developments in microfluidics have brought forth a variety of new devices that can potentially revolutionize traditional biomedical and chemical experiments. Examples of microfluidic devices can be found in Fu, A. Y., et al., “A microfabricated fluorescence-activated cell sorter, Nature Biotechnology,” 1999, 17(11), pp. 1109-1111, Tai, Y. C., et al., “Integrated micro/nano fluidics for mass-spectrometry protein analysis,” International Journal of Nonlinear Sciences and Numerical Simulation, 2002, 3(3-4), pp. 739-741, Tokeshi, M., et al., “Chemical processing on microchips for analysis, synthesis, and bioassay,” Electrophoresis, 2003, 24(21): pp. 3583-3594, Doyle, P. S., et al., “Self-assembled magnetic matrices for DNA separation chips,” Science, 2002, 295(5563), pp. 2237-2237, Trau, D., et al., “Genotyping on a complementary metal oxide semiconductor silicon polymerase chain reaction chip with integrated DNA microarray, Analytical Chemistry,” 2002, 74(13), pp. 3168-3173, and Liu, S. R., “A microfabricated hybrid device for DNA sequencing,” Electrophoresis, 2003, 24(21), pp. 3755-3761, which are hereby incorporated by reference in their entirety for all purposes. Another such device is the optofluidic microscope (OFM) described in U.S. patent application Ser. No. 11/686,095, filed on Mar. 14, 2007, by Changhuei Yang and Demetri Psaltis, entitled OPTOFLUIDIC MICROSCOPE DEVICE, which is hereby incorporated by reference in its entirety for all purposes. The OFM fuses the advantage of optical imaging in providing high resolution and the advantages of microfluidics, such as low cost and high throughput. Further, OFM's application in nematode imaging and phenotyping has been reported in Heng, X., et al., “Optofluidic microscope, a miniature microscope on a chip,” 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences (μTAS), 2005, which is hereby incorporated by reference in its entirety for all purposes.
FIGS. 1A and 1B are schematic drawings an OFM device having a body that forms a fluid channel. An object is passing through the fluid channel generally in the flow direction. The body includes an aperture layer having an aperture array of light transmissive regions (e.g., apertures, slits, etc.). The aperture layer is located adjacent to the light detector layer having an array of light detecting elements. The aperture array is oriented at a small angle β relative to the fluid channel (FIG. 1B). As an object passes over the aperture array, light detecting elements detect light through the light transmissive regions. The sensor of a processor communicating with the sensor generates time-varying data in the form of a line scan which can be compiled into an image of the object.
FIG. 2A illustrates an image of wild-type C. elegans larvae at the first larval stage that was generated by an OFM device. FIG. 2B is an OFM generated image of a dpy-24 mutant that was generated by an OFM device. FIG. 2C illustrates the aspect ratio of wild-type larvae and dpy-24 mutants that was generated by an OFM device.
In many cases, the light transmissive regions in the OFM device may be of a small size due to the compactness of the OFM device. In these cases, light detector may detect a weak signal. The reduced optical transmission through small apertures is described in Bouwkamp, C. J., “Diffraction theory,” Reports on Progress in Physics XVIII, 1954, p. 35 and de Abajo, F., “Light transmission through a single cylindrical hole in a metallic film,” Optics Express, 2002, 10(25), pp. 1475-1484, which are hereby incorporated by reference in their entirety for all purposes. The weak transmission signal can be buried in noise. Although strong illumination from strong sources such as powerful lasers can help to increase the total transmission through small light transmissive regions, high-intensity light may also have adverse effects on the biological specimens.
Darkfield Imaging
The ability of an optical sensor to detect light signals, especially weak light signals, can be limited by the presence of a bright background. This limitation is described in R. Narayanaswamy and O. Wolfbeis, “Optical sensors: industrial, environmental and diagnostic applications,” Springer Berlin, 2004, and G. C. Cox, “Optical imaging techniques in cell biology,” Boca Raton: CRC/Taylor & Francis, 2007, which are hereby incorporated by reference in their entirity for all purposes. As such, pre-detection background suppression providing a darkfield can be important in the detection of light signals. The benefit of a darkfield image can be appreciated by drawing an analogy to the visibility of stars in the night and their apparent absence during the day—the absence of the bright background can significantly enhance the relative contrast of the light fields.
Darkfield imaging has numerous advantages and applications. For example, darkfield imaging of biological specimens can be advantageous because the outlines of specimens tend to show up prominently in darkfield images as bright lines delineating the objects and because the interior structures of specimens show up well for similar reasons. In some cases, the increased contrast provided by darkfield imaging can eliminate the need for staining specimens, which could be vitally useful for certain time critical medical procedures such as pathological examination of resected tissue samples during surgery.
Prior darkfield imaging devices such as conventional darkfield microscopes use complex and expensive components to generate a darkfield image. For example, FIG. 3A is a schematic drawing of portions of a conventional darkfield microscope having a cardioid darkfield condenser. The condenser is structured so that light emerging from is incident at large angles on the object. The objective on the opposite side of the object is selected so that the numerical aperture is sufficiently small to ensure that the objective will not collect the illumination light field in the absence of the object. The optics process the scattered or diffracted light due to the presence of the object, to render a darkfield image. FIG. 3B is a darkfield image of Chlamydomonas generated using the conventional darkfield microscope. FIG. 3B is a bright field image of Chlamydomonas generated using a bright field microscope.
Conventional darkfield microscopes are also difficult to operate and can require extensive training. Since the illumination light field must be excluded from the collection aperture of the objective, it precludes the use of high numerical aperture objectives. Additionally, using conventional darkfield microscopes requires familiarity with their working principles, and the positioning of condenser, sample, and objective demands precision.